A. Hypothesis: Factors regulating PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, and TNAP, are involved in root formation and cementogenesis. The findings to date demonstrate the importance of regulators of Pi/PPi and of functional SIBLING family genes/proteins during mineralization, and highlight the importance of fine tuning physiochemical and cellular molecular factors toward achieving a homeostatic balance required for formation and for regeneration of periodontal tissues. 1. Teeth obtained from Alpl KO mice, beyond the known cementum phenotype (no acellular cementum, altered PDL and exfoliation of teeth due to lack of PDL attachment to the root surface in humans and mice) exhibit a marked dentin and enamel phenotype also seen in humans. Further, these defects were corrected by TNAP enzyme replacement in mice and, in clinical trials, in humans. 2. ChimAP, a novel treatment for hypophosphatasia (HPP), intestinal-like chimeric alkaline phosphatase (ChimAP), improved skeletal mineralization in Alpl-/- mice. 3. A novel mouse model for tooth-specific HPP (odonto-HPP) was created by knocking in an autosomal dominant ALPL missense mutation (causing a base change, A116T), which was identified in a human kindred. Alpl+/A116T mice featured no detectable changes in long bone properties, but exhibited alterations in the alveolar bone, including radiolucencies and resorptive lesions, osteoid accumulation on the alveolar bone crest, and significant differences in several bone properties measured by microCT. 4. Phospho1 KO and AlplxPhospho1 dKO: Teeth obtained from AlplxPhospho1 dKO mice exhibit a more profound dentin phenotype than those comparable tissues obtained from KO of either Alpl or Phospho1, demonstrating the important function of PHOSPHO1 in controlling Pi levels in matrix vesicles (MVs), synergistically with TNAP. 5. ANK, NPP1 and TNAP in vivo and vitro: Examining Ank and Npp1 KO animals, demonstrated a compensatory mechanism related to gene expression. Cementum analysis of tissues obtained from either Npp1 or Ank KO mice (i.e., decreased PPi locally) exhibited a marked increase in protein expression for two SIBLINGS, OPN and DMP1, and further, cementoblasts exposed to Pi in vitro also exhibited an increase in transcripts for these two genes. We noted cementoblasts in vitro expressed Alpl gene and TNAP protein early, whereas induction of Enpp1 gene/protein expression required mineralization conditions, similar to in vivo findings. These patterns were confirmed in human teeth where rich TNAP expression in the PDL is contrasted by specific localization of NPP1 to root-lining cementoblasts. These studies clarify the expression patterns and functions of TNAP and NPP1 in cementum mineralization, suggesting that the early expression of TNAP creates a low PPi environment allowing for AEFC initiation, while later expression of NPP1 increases PPi, restricting AEFC apposition. These results highlighted the importance of Pi/PPi modulation for cementogenesis and provide the rationale for our ongoing in vivo studies using periodontal wound healing models in rodents to deliver factors controlling Pi/PPi, both locally at the defect site as well as systemically. B. Hypothesis: Hormones involved in regulating mineral metabolism e.g., PTH, 1,25D, and FGF23 modulate the activity of DMP-1, a key extracellular matrix protein belong to the SIBLING family. Murine cementoblasts (OCCM-30), osteocyte-like cells (MLO-Y4, and MLO-A5) known to express Dmp1 were used to analyze effects of PTH and 1,25D in regulating DMP1 as well as other factors involved in mineral homeostasis, by employing RNA-seq. Collectively, the results suggest that both PTH and 1,25D share complementary effects toward maintaining mineral homeostasis by partly regulating similar genes/proteins associated with calcium and phosphate metabolism, while also exerting distinct roles on factors modulating mineral metabolism. Regulating genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health. C. Hypothesis: Extracellular matrix proteins, especially those within the SIBLING family, play a vital role in development and maintenance of the periodontium. We have focused on determining the mechanism for lack of cementum formation in Bsp null mice using in vivo assays, (TEM/SEM; IHC, in situ hybridization and special stains for collagen and mineral deposition), in vitro assays, (using Bsp null cell functional analysis and RNA-Seq analysis from freshly isolated PDL tissues and OCCM cells with BSP KO). While healthy wild-type (WT) mice exhibit a strong integration of Sharpeys fibers at the periodontal ligament (PDL)-root surface, Bsp-/- mice feature a lack of cementum ultrastructure, loss of Sharpeys fibers integration, and detachment of PDL from the root surface. In a closely related project in the laboratory of our collaborator, Dr. Harvey Goldberg, the effect of soft diet on incisor malocclusion and periodontal destruction in Bsp-/- mice was used to better understand the role of BSP in wider periodontal function. Bsp-/- mice in the soft diet group featured normal body weight, long-bone length, and serum ALP activity, suggesting that tooth dysfunction and malnutrition contribute to growth and skeletal defects reported in Bsp-/- mice. Bsp-/- incisors erupted at a slower rate, which likely leads to the observed thickened dentin and enhanced mineralization of dentin and enamel towards the apical end. We propose that the decrease in eruption rate is due to lack of acellular cementum and associated defective periodontal attachment, noted in both soft and hard diet groups. These data demonstrate the importance of BSP in maintaining proper periodontal function and alveolar bone remodeling, and point to dental dysfunction as a causative factor of skeletal defects observed in Bsp-/- mice. D. To determine the effect of disorders associated with collagen function on periodontal tissues. Logic would suggest that individuals with osteogenesis imperfect (O.I.) would have marked periodontal defects. Yet, reports to date focus on general bone pathology and in some cases dentin defects (dentinogenesis imperfecta (D.I.)), and reports to date investigating periodontal/cementum defects are limited. Our studies show, compared to controls, AD Brtl+/- mice featured thinner dentin, ectopic pulp calcification and alveolar bone defects, while Gly610Cys+/- mice had relatively normal morphology at young ages. AR models exhibited significant periodontal changes. Both Crtap-/- and Ppib-/- mice featured parallel phenotypes including increased acellular cementum, and reduced cellular cementum, dentin width and root length. For the first time, we identify periodontal changes in mice resulting from AR and AD OI. Changes reflect developmental defects, as well as altered function and remodeling during later stages in life. These findings will provide insights into oral-dental health of human subjects with OI.